The pharmacological treatment of pain has long been limited by the negative side effects of opioids: development of tolerance, dependence, and possibility of overdose. A literature has developed linking opiate side effects to their influence on glial cells of the central nervous system (CNS). Herein lies a proposal to investigate the role of an opiate-mediated glial activation, via the signaling pathway mediated by toll-like receptor 4 (TLR4). TLR4, an integral membrane receptor expressed in glia but no in neurons within the CNS, functions in complex with its accessory protein, Myeloid Differentiation protein-2 (MD-2). The TLR4/MD-2 complex is crucial to the TLR4-signaling transduction. The proposal's central hypothesis is that inhibition of the TLR4/MD-2 association will impede the TLR4-signaling pathway, thereby preventing the negative side effects from opiate-induced glial activation. By selectively blocking the critical protein-protein interactions between TLR4 and MD-2, opiate tolerance and dependence is predicted to attenuate, thereby increasing the efficacy of current pain pharmacotherapies. This approach is innovative, as it is the first proposal aimed at the inhibition of glial- mediated opioid side effects. Further, the research is expected to yield significant outcomes: (1) inhibitors of the TLR4/MD-2 interaction will be prototypes for the development of drugs to counteract opioid side effects from tolerance to addiction and overdose. The TLR4/MD-2 interaction is also implicated in other pathologies (e.g. sepsis), and will therefore serve potential targets for various diseases. (2) Importantly, antagonists of the TLR4/MD-2 interaction will elucidate the contribution of the TLR4 pathway to opiate-induced glial activation. The inhibitors will help determine the mechanism of action of the TLR4 pathway itself, shedding light on the molecular specificity with which TLR4 recognizes its ligands. The proposed studies are built on a strong collaborative team with a spectrum of expertise covering from protein design, biochemistry and biophysical assay development, x-ray structural analysis, and animal models for pain management. The proposed studies employ computationally designed peptides derived from the TLR4-bidning regions of MD-2, which is expected to compete with the full-length MD-2 protein and prevent the TLR4 signal transduction. These peptides will provide starting points for the small-molecule inhibitors. The significance of this work lies in its impacts on both clinical and scientific advancement. Dissecting the mechanism of opiate-induced glial activation will help us understand the development of opioid tolerance and addiction, as well as establish a novel angle from which to address drug dependence and abusing of these opiates. Regarding its impact on scientific advancement, the proposed studies will illuminate the molecular mechanism of TLR4 activation, which is relevant to a many interrelated signaling and immunomodulatory pathways, and crucial to the understanding of pain suppression. PUBLIC HEALTH RELEVANCE: The proposed research aims to unravel the mechanism of opioid-induced glial activation that both hinders the ability of opioids to effectively control pain and also importantly contributes to the development of drug addiction and abuse. State-of-the-art technologies will be employed to define, design, create, and test new chemical entities predicted to prevent opioid induced glial activation, thereby optimizing opioid analgesia while preventing negative consequences of clinical opioid use.